Purpose :
The efficient intracellular delivery of functional proteins into retinal cells is needed to fully realize the potential of macromolecular therapeutics in treating a variety of retinal diseases. Recent discoveries revealed a novel approach of protein delivery that can potently deliver nanomolar concentrations of negatively supercharged protein complex into mammalian cells both in vitro and in vivo. We propose to validate the potential of this new cationic lipid-mediated approach to deliver functional protein into mouse retinal cells.

Methods :
We used a Cre recombinanase reporter mouse model with a floxed-stop-tdTomato reporter allele in the ROSA26 locus to test this protein delivery system. The tdTomato gene turns on in the cells expressing Cre recombinase following Cre-mediated excision of a loxP-flanked transcriptional "stop" sequence. We translationally fused a super-negatively charged GFP variant (–30)GFP to Cre recombinase to generate (–30)GFP-Cre and then complexed it with a variety of commercially available or customized cationic lipids.The protein-lipid complex was delivered into the retinas of P0-P2 mice by sub-retinal injection. The delivery efficiency of different protein concentration and/or variant cationic lipids was evaluated by assessing the expression level of TdTomato in the retinas using fluorescent microscopy.

Results :
TdTomato expression was oberserved in the retinas injected with (-30)GFP-Cre protein complexed with cationic lipids. Lipofectamine 2000 resulted in highest delivery efficiency of the functional (–30)GFP-Cre over a range of concentrations (20-200 nM). Customized lipids D23, 80-O14B 16:2:2:1, and EC16-63 16:2:1:1 also resulted in moderate functional (–30)GFP-Cre delivery. The TdTomato expression was found to be at higher level in the RPE and Muller cells than that in the inner nuclear layers and outer nuclear layers of the retina.

Conclusions :
Our proof-of-concept experiments indicate that cationic lipids are able to complex with and effectively deliver negatively charged proteins into mouse retinal cells. This should serve as a basis for designing novel systems for a controlled and scalable delivery of the therapeutic proteins, including genome-editing proteins, into the retinal cells, with promising implications for the treatment of retinal diseases affecting all kinds of cells.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.